In general, cellular metabolism consists of the production of major precursors and energy by oxidative and catabolic reactions of nutrients input from the outside, and synthesis of biomolecules using the major precursors and energy. The constituents of the bioenergy generated in this process and consumed in the synthesis process are electrons and protons, and coenzymes, NADP(H) and NAD(H), act as the main carrier.
NADP(H) and NAD(H) are essential elements in vivo, and oxidized forms (NAD+ and NADP+) are regenerated (NADH and NADPH) in the central pathway. The ratio between the oxidation and reduction form is also controlled through a regenerative pathway (salvage pathway) that is able to be interconverted between oxidation/reduction forms.
The oxidation/reduction ratio of the coenzymes in cells can be used as an important indicator of bioactivity. The quantification of NADP(H) and NAD(H) may be used to determine whether normal physiology in vivo is maintained, metabolism abnormality occurs, and the like. This may be an important indicator for determining the presence of cells, cell viability, and food contamination in samples, and may be widely applied to other fields as well.
In order to utilize quantitative determination of the coenzymes, various coenzyme measurement methods are being developed. As a representative method, there is a method of measuring absorbance or fluorescence using intrinsic wavelength of NADP(H) or NAD(H), but since the absorbance and the fluorescence value of the coenzymes are quite low, there is a disadvantage in that a relatively accurate measurement value is obtainable only in a defined (artificial) reaction composition, and thus it is difficult to apply this method directly to various kinds of natural samples.
Other methods for quantitative determination of coenzymes are also established. However, it is difficult to separately measure NADP+/NADPH and NAD+/NADH, which are oxidation/reduction forms of coenzyme, and it is difficult to perform the measurement effectively when a concentration of coenzyme is very low. For example, NADP(H) and NAD(H) have similar absorbance and fluorescence spectrums, and thus it is difficult to distinguish the NADP(H) and NAD(H). When an amount of the sample is small, the signal to noise ratio (S/N ratio) is low, and thus it is very difficult to distinguish the NADP(H) and NAD(H). In addition, since oxidized NADP+ has relatively poor optical properties, it is not easy to measure the coenzyme having the corresponding form.
In order to overcome problems known in the related art, a method of using high performance liquid chromatography (HPLC), a method of using a coupling reaction including dehydrogenase which induces coenzyme reduction through oxidation of a specific substrate, or a method of using an artificial fluorescent substance, or the like, has been developed. However, these methods require a multi-step pretreatment protocol, and these methods are not able to be used for direct real-time observation of the coenzyme concentration gradient in the body or for quantitative analysis.
In order to solve the above-described disadvantages, a method using molecular imaging equipment has been developed. A confocal laser scanning microscope is used to observe an intrinsic fluorescence wavelength of the coenzyme with high resolution with a high power continuous wave (CW) laser. Alternatively, two photon microscopy using two photons minimizes cell damage or photobleaching according to single radiation of a high energy wavelength due to two successive radiations with low energy level in an image acquisition process, and thus the measurement of the coenzyme is improved. However, the above method is still a fluorescent measurement method using the intrinsic wavelength of NADPH, and it is difficult to perform accurate quantitative analysis or distinguish NADPH from NADH because the S/N ratio is low, and thus another solution is urgently required.